Prevotella histicola preparations and the treatment of autoimmune conditions

ABSTRACT

This document provides methods and materials related to  Prevotella histicola  preparations. For example,  Prevotella histicola  preparations in the form of an oral medicament or dietary supplement (e.g., a pill, tablet, capsule) are provided. In addition, methods and materials for using a  Prevotella histicola  preparation provided herein as an anti-inflammatory agent are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/385,554, filed Dec. 20, 2016, which is a continuation of U.S.application Ser. No. 14/643,156, filed Mar. 10, 2015 (now U.S. Pat. No.9,555,066), which is a continuation of U.S. application Ser. No.14/086,090, filed Nov. 21, 2013 (now U.S. Pat. No. 9,005,603), which isa continuation of U.S. application Ser. No. 13/505,169 (now U.S. Pat.No. 8,617,536), filed Apr. 30, 2012, which is a National Stageapplication under 35 U.S.C. §371 and claims benefit under 35 U.S.C.§119(a) of International Application No. PCT/US2010/054314, having anInternational Filing Date of Oct. 27, 2010, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 61/299,068, filed onJan. 28, 2010 and U.S. Provisional Application Ser. No. 61/256,731,filed on Oct. 30, 2009. The disclosures of the prior applications areconsidered part of (and are incorporated by reference in) the disclosureof this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DK071003 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND 1. Technical Field

This document relates to Prevotella histicola preparations and the useof Prevotella histicola preparations to treat autoimmune conditions(e.g., arthritis and multiple sclerosis).

2. Background Information

A large reservoir of microorganisms lives in the digestive tracts ofanimals and is often referred to as the gut flora or microflora.Bacteria make up most of the flora in the colon and about 60 percent ofthe dry mass of feces. In fact, between 300 and 1000 different speciesmay live in the gut.

SUMMARY

This document provides methods and materials related to Prevotellahisticola preparations. For example, this document provides Prevotellahisticola preparations in the form of an oral medicament or dietarysupplement (e.g., a pill, tablet, capsule). In addition, this documentprovides methods for using a Prevotella histicola preparation providedherein as an anti-inflammatory agent. In some cases, a Prevotellahisticola preparation provided herein can be used as an oralanti-inflammatory medicament or dietary supplement to treat autoimmuneconditions such as arthritis and multiple sclerosis. The Prevotellahisticola preparations provided herein can contain live or killedPrevotella histicola microorganisms. In some cases, a compositionincluding, or consisting essentially of, a culture supernatant from aPrevotella histicola culture can be used as described herein. Forexample, a culture supernatant from a Prevotella histicola culture canbe administered (e.g., orally administered) to a mammal to treat anautoimmune condition or inflammatory condition in the mammal. Such aculture supernatant can include live or killed Prevotella histicolamicroorganisms. In some cases, a culture supernatant from a Prevotellahisticola culture can lack live Prevotella histicola microorganisms. Insome cases, a culture supernatant from a Prevotella histicola culturecan include lysed Prevotella histicola microorganisms. Any appropriatemedia can be used to culture Prevotella histicola microorganisms to forma culture supernatant. For example, broth (e.g., trypticase soy broth)can be used to culture Prevotella histicola microorganisms to form aculture supernatant.

The Prevotella histicola preparations and compositions provided hereinand the methods for using the Prevotella histicola preparations andcompositions provided herein can allow medical professionals to treatmammals (e.g., human patients) suffering from an autoimmune condition.In some cases, the methods and materials provided herein can allowhumans to supplement their diets with bacterial organisms having theability to reduce the severity or development of an autoimmunecondition.

In general, one aspect of this document features a method for treatingan autoimmune condition in a mammal. The method comprises, or consistsessentially of, administering a composition comprising, or consistingessentially of, live Prevotella histicola to the mammal under conditionswherein the severity of the autoimmune condition is reduced. The mammalcan be a human. The autoimmune condition can be multiple sclerosis. Theautoimmune condition can be arthritis. The administering step cancomprise an oral administration. The composition can be a pill, tablet,or capsule. The composition can be a pill, tablet, or capsule configuredto deliver the live Prevotella histicola to the intestines of themammal. The severity of the autoimmune condition can be reduced bygreater than about 25 percent following the administering step. Theseverity of the autoimmune condition can be reduced by greater thanabout 50 percent following the administering step. The severity of theautoimmune condition can be reduced by greater than about 75 percentfollowing the administering step. The method can comprise identifyingthe mammal as having the autoimmune condition prior to theadministration. Representative cells of the Prevotella histicola can bethe cells deposited as NRRL accession number B-50329.

In another aspect, this document features a nutritional supplementcomprising, or consisting essentially of, live Prevotella histicola. ThePrevotella histicola can be encapsulated to be released in the intestineof a mammal. Representative cells of the Prevotella histicola can be thecells deposited as NRRL accession number B-50329.

In another aspect, this document features a human food productsupplemented with live Prevotella histicola. The product can containbetween 1×10⁷ to 1×10¹¹ cells of the Prevotella histicola. The productcan be selected from the group consisting of milk, yogurt, milk powder,tea, juice, cookies, wafers, crackers, and cereals.

In another aspect, this document features a method for treating anautoimmune condition in a mammal. The method comprises, or consistsessentially of, administering a composition comprising, or consistingessentially of, dead Prevotella histicola to the mammal under conditionswherein the severity of the autoimmune condition is reduced. The mammalcan be a human. The autoimmune condition can be multiple sclerosis. Theautoimmune condition can be arthritis. The administering step cancomprise an oral administration. The composition can be a pill, tablet,or capsule. The composition can be a pill, tablet, or capsule configuredto deliver the dead Prevotella histicola to the intestines of themammal. The severity of the autoimmune condition can be reduced bygreater than about 25 percent following the administering step. Theseverity of the autoimmune condition can be reduced by greater thanabout 50 percent following the administering step. The severity of theautoimmune condition can be reduced by greater than about 75 percentfollowing the administering step. The method can comprise identifyingthe mammal as having the autoimmune condition prior to theadministration. Representative cells of the Prevotella histicola can bethe cells deposited as NRRL accession number B-50329.

In another aspect, this document features a nutritional supplementcomprising, or consisting essentially of, dead Prevotella histicola. ThePrevotella histicola can be encapsulated to be released in the intestineof a mammal. Representative cells of the Prevotella histicola can be thecells deposited as NRRL accession number B-50329.

In another aspect, this document features a human food productsupplemented with dead Prevotella histicola. The product can containbetween 1×10⁷ to 1×10¹¹ cells of the Prevotella histicola. The productcan be selected from the group consisting of milk, yogurt, milk powder,tea, juice, cookies, wafers, crackers, and cereals.

In another aspect, this document features a method for treating anautoimmune condition in a mammal. The method comprises, or consistsessentially of, administering a composition comprising, or consistingessentially of, a culture supernatant from a Prevotella histicolaculture to the mammal under conditions wherein the severity of theautoimmune condition is reduced. The mammal can be a human. Theautoimmune condition can be multiple sclerosis. The autoimmune conditioncan be arthritis. The administering step can comprise an oraladministration. The composition can be a pill, tablet, or capsule. Thecomposition can be a pill, tablet, or capsule configured to deliver theculture supernatant to the intestines of the mammal. The severity of theautoimmune condition can be reduced by greater than about 25 percentfollowing the administering step. The severity of the autoimmunecondition can be reduced by greater than about 50 percent following theadministering step. The severity of the autoimmune condition can bereduced by greater than about 75 percent following the administeringstep. The method can comprise identifying the mammal as having theautoimmune condition prior to the administration. Representative cellsof the Prevotella histicola can be the cells deposited as NRRL accessionnumber B-50329.

In another aspect, this document features a nutritional supplementcomprising, or consisting essentially of, a culture supernatant from aPrevotella histicola culture. The culture supernatant can beencapsulated to be released in the intestine of a mammal. Representativecells of the Prevotella histicola can be the cells deposited as NRRLaccession number B-50329.

In another aspect, this document features a human food productsupplemented with a culture supernatant from a Prevotella histicolaculture. The culture supernatant can be obtained from a culture havinggreater than 1×10³ (e.g., greater than 1×10³, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, or 1×10¹¹) Prevotella histicola cells permL of media. The product can be selected from the group consisting ofmilk, yogurt, milk powder, tea, juice, cookies, wafers, crackers, andcereals.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Modulation of EAE by Prevotella histicola. Treatment with P.histicola protected a majority of DR3DQ8 mice from developing EAE, whilemedium fed control mice or DR3DQ8 mice treated with control commensalbacteria (Capnocytophagia sputigena or E. coli) exhibited 100% diseaseincidence, thereby indicating that P. histicola have an immunomodulatoryeffect. HLA-DR3DQ8 or DQ8 control Tg mice were immunized with PLP₉₁₋₁₁₀myelin antigen emulsified in CFA. Pertussis toxin was given at day 0 and2 post-immunization. Seven days post immunization mice were gavaged onalternate days either with P. histicola or Capnocytophagia sputigena orE. coli (2×10⁹ CFU in 100 μL of trypticase soy broth (TSB) culturemedia) or medium for 7 doses. Mice were monitored daily for developmentof EAE and scored using standard EAE scoring criteria as describedelsewhere (Mangalam et al., J. Immunol., 182(8):5131-9 (2009)). At theend of treatment, tissue and sera were collected from the mice forfurther analysis. No immuno-modulatory effect was observed with othertested human or mouse commensal bacteria.

FIG. 2. P. histicola treated DR3DQ8 mice exhibited reduced PLP₉₁₋₁₁₀specific T cell proliferation as compared to sham treated mice.Splenocytes were collected from mice immunized with PLP and treated withP. histicola or medium (sham) and were stimulated in vitro with thePLP₉₁₋₁₁₀ polypeptide.

FIG. 3. P. histicola treated DR3DQ8 mice exhibited reduced level ofIL-17 and increased levels of IL-10 as compared to sham treated mice.Levels of IFN-γ were not different between the two groups of mice.

FIGS. 4A-B. FIG. 4A P. histicola treated DR3DQ8 mice exhibited reducedlevels of MIP-1α, MIP-1β, and MCP-1 in splenocytes as compared to levelsmeasured in splenocytes from sham treated mice. FIG. 4B Levels of thesechemokines were higher in splenocytes from P. histicola treated mice ascompared to levels measured in splenocytes from sham treated mice.

FIG. 5. Modulation of collagen-induced arthritis (CIA) by Prevotellahisticola. Immunization of HLA-DQ8.AEo Tg mice with type II collagen(CII) leads to development of collagen-induced arthritis, a model forrheumatoid arthritis (Taneja et al., J. Immunol., 56:69-78 (2007)).Treatment with P. histicola protected DQ8.AEo mice from developingarthritis. Three groups of mice were included: (1) mice immunized withCII/CFA and treated with medium, (2) mice immunized with CII and treatedwith P. histicola, and (3) a control group, which included miceimmunized with P. histicola without CII. Ten days post immunization,mice were gavaged three times per week with P. histicola (2×10⁹ CFU in100 μL of TSB culture media) for up to 7 weeks. Group 1 and group 2 micewere boosted with CII/IFA in the 6^(th) week. Mice were monitored forarthritis for up to 10 weeks. Mice immunized with CII and treated withP. histicola exhibited a dramatic decrease in disease incidence as wellas milder disease. Mice only given P. histicola did not develop anyarthritis.

FIGS. 6A-B. P. histicola treated mice exhibited reduced humoralantigen-specific response. FIG. 6A Anti-CII antibodies in sera collectedbefore and after treatment with P. histicola exhibited a reduction inantibodies. Anti-CII antibodies were tested by ELISA. FIG. 6B T cellproliferation to CII in vitro did not exhibit any significant reductionin P. histicola treated mice. Only P. histicola gavaged mice did notexhibit any antigen-specific response.

FIG. 7. All tested proinflammatory and immunomodulatory cytokinesproduced in response to CII were reduced in P. histicola treated micecompared to medium fed (control) mice. Cytokines were measured fromserum of mice by using a multiplex array system.

FIG. 8. Mice treated with P. histicola exhibited higher numbers ofCD4⁺GITR⁺ T regulatory cells and CD11c⁺CD103⁺ dendritic cells insplenocytes. The FACs analysis after staining with conjugated antibodiesis shown.

FIG. 9. Treatment with P. histicola leads to an increase in regulatorydendritic cells, CD11c⁺CD103⁺, in lamina propria.

FIG. 10 is a sequence comparison of 16S rRNA nucleic acid from P.histicola deposited with the ARS Culture Collection (NRRL accessionnumber B 50329, deposited Oct. 28, 2009) and set forth as Query (SEQ IDNO:1) to 16S rRNA nucleic acid from GenBank® accession number EU126662.1Prevotella histicola strain N12-20 (GI No.:

157366663) set forth as subject (Sbjct; SEQ ID NO:2).

FIG. 11 contains flow cytometry graphs plotting the percentage ofCD4⁺FoxP3⁻ cells in spleen or mesenteric lymph nodes from sham treatedmice or P. histicola treated mice.

FIG. 12 contains flow cytometry histograms plotting the percentage ofCD11b⁺ CD11c⁺CD103⁺ dendritic cells (DCs) in spleen or mesenteric lymphnodes from sham treated mice or P. histicola treated mice.

FIG. 13A is a graph plotting the level of proliferation (Δ cpm) for DCsisolated from sham treated or P. histicola treated mice and culturedwith CD4⁺ cells isolated from sham treated or P. histicola treated micein the presence of CII. FIG. 13B is a graph plotting the levels of IL-10and IL-17 (pg/nL) from CII containing cultures of either DCs isolatedfrom sham treated or P. histicola treated mice in combination witheither CD4⁺ cells isolated from sham treated or P. histicola treatedmice. The ratio of DCs to CD4⁺ cells was 1:1. S represents sham treated,and P. hist. represents P. histicola treated.

FIG. 14 contains flow cytometry graphs plotting the percentage ofCD4⁺CD25⁺FoxP3⁺ cells in spleen or lamina propria from sham treated miceor P. histicola treated mice.

FIG. 15 is a graph plotting the average clinical score of EAE for micetreated with media allow (Med fed), P. histicola (P. histicola fed), orculture supernatant from a P. histicola culture (P. histicola CultureSupernatant fed).

DETAILED DESCRIPTION

This document provides methods and materials related to P. histicolapreparations. For example, this document provides compositionscontaining P. histicola (e.g., live P. histicola, killed P. histicola,P. histicola components, or lysed P. histicola). Such compositions cancontain any amount of P. histicola or P. histicola components. In somecases, a composition provided herein can contain P. histicola (e.g.,live or killed P. histicola) or P. histicola components in an amountsuch that between 0.001 and 100 percent (e.g., between 1 and 95 percent,between 10 and 95 percent, between 25 and 95 percent, between 50 and 95percent, between 20 and 80 percent, between 50 and 95 percent, between60 and 95 percent, between 70 and 95 percent, between 80 and 95 percent,between 90 and 95 percent, between 95 and 99 percent, between 50 and 100percent, between 60 and 100 percent, between 70 and 100 percent, between80 and 100 percent, between 90 and 100 percent, or between 95 and 100percent), by weight, of the composition can be P. histicola or P.histicola components. In some cases, a composition provided herein cancontain between about 10³ and 10⁸ live P. histicola microorganisms.

In some cases, a composition provided herein can contain P. histicola(e.g., live P. histicola microorganisms) in the amounts and dosages asdescribed elsewhere for probiotic bacteria (U.S. Patent ApplicationPublication No. 2008/0241226; see, e.g., paragraphs [0049-0103]). Inaddition, a composition provided herein containing P. histicola (e.g.,live P. histicola microorganisms) can be administered as describedelsewhere for probiotic bacteria (U.S. Patent Application PublicationNo. 2008/0241226; see, e.g., paragraphs [0049-0103]).

Live P. histicola microorganisms can be obtained from the digestivesystem of any appropriate mammal (e.g., a human). For example, P.histicola microorganisms can be isolated from small intestinal mucosa(e.g., a small bowel biopsy or aspirate sample) of a human (e.g., ahuman patient diagnosed with celiac disease). P. histicola strains canbe identified via 16S rRNA PCR using standard 16S rRNA primers. The 16SrRNA sequence used to identify P. histicola can be as set forth in FIG.10. In some cases, P. histicola microorganisms can be obtained from theARS Culture Collection (NRRL accession number NRRL B-50329, depositedOct. 28, 2009).

Any appropriate method can be used to obtain a culture of P. histicolamicroorganisms. For example, standard microbial culturing techniques canbe used to obtain P. histicola or P. histicola components. In general,P. histicola microorganisms can be cultured in broth containing milk(e.g., skim milk) to obtain a culture containing greater than 1×10⁸ P.histicola per mL of broth. The P. histicola microorganisms can beremoved from the broth via centrifugation. Once obtained, the live P.histicola microorganisms can be formulated into a medicament ornutritional supplement composition for administration to a mammal (e.g.,a human), can be added to a food product for consumption, or can befrozen for later use. In some cases, the obtained P. histicolamicroorganisms can be treated (e.g., chemical treatment, repeatedfreeze-thaw cycles, antibiotic treatment, or fixation treatment such aformalin treatment) to obtain a composition of killed or lysed P.histicola microorganisms or can be processed (e.g., lysed followed byfractionation) to obtain a composition of P. histicola components.

In some cases, a P. histicola preparation, which can be stored frozen in2× skim milk, can be thawed and grown on CDC Anaerobe Laked Sheep BloodAgar with kanamycin and vancomycin (KV) (Becton, Dickson and Company,Sparks, Md., product number 221846) in an anaerobe jar with AnaeroPackSystem (product number 10-01, Mitsubishi Gas Chemical America, Inc., NewYork, N.Y.). The culture can be incubated at 35-37° C. for at least 48hours.

A composition containing P. histicola or P. histicola components can bein the form of an oral medicament or nutritional supplement. Forexample, compositions containing P. histicola or P. histicola componentscan be in the form of a pill, tablet, powder, liquid, or capsule.Tablets or capsules can be prepared by conventional means withpharmaceutically acceptable excipients such as binding agents, fillers,lubricants, disintegrants, or wetting agents. The tablets can be coatedby methods known in the art. In some cases, a composition containing P.histicola or P. histicola components can be formulated such that live orkilled P. histicola or P. histicola components are encapsulated forrelease within the intestines of a mammal. Liquid preparations for oraladministration can take the form of, for example, solutions, syrups, orsuspension, or they can be presented as a dry product for constitutionwith saline or other suitable liquid vehicle before use. In some cases,a composition provided herein containing P. histicola (e.g., live P.histicola microorganisms) can be in a dosage form as described elsewhere(U.S. Patent Application Publication No. 2008/0241226; see, e.g.,paragraphs [0129-0135]). For example, a composition provided herein canbe in the form of a food product formulated to contain P. histicola(e.g., live P. histicola microorganisms) or P. histicola components.Examples of such food products include, without limitation, milk (e.g.,acidified milk), yogurt, milk powder, tea, juice, beverages, candies,chocolates, chewable bars, cookies, wafers, crackers, cereals, treats,and combinations thereof.

A composition containing P. histicola or P. histicola components cancontain other ingredients such as buffers, radical scavengers,antioxidants, reducing agents, or mixtures thereof. For example, acomposition containing live P. histicola can be formulated to containbotanicals, vitamins, minerals, or combinations thereof. In some cases,a composition provided herein containing P. histicola (e.g., live P.histicola microorganisms) can contain other ingredients as describedelsewhere (U.S. Patent Application Publication No. 2008/0241226; see,e.g., paragraphs [0104-0128]).

In some cases, a composition containing P. histicola or P. histicolacomponents can contain a pharmaceutically acceptable carrier foradministration to a mammal, including, without limitation, sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and organic esters. Aqueouscarriers include, without limitation, water, alcohol, saline, andbuffered solutions. Pharmaceutically acceptable carriers also caninclude physiologically acceptable aqueous vehicles (e.g., physiologicalsaline) or other known carriers for oral administration.

This document also provides methods and materials for using acomposition containing P. histicola or P. histicola components as ananti-inflammatory agent. In some cases, a composition containing P.histicola or P. histicola components can be used to treat autoimmuneconditions such as arthritis, multiple sclerosis, systemic lupuserythematosus (SLE), type 1 diabetes (T1D), and Crohn's disease. In somecases, a composition containing P. histicola or P. histicola componentscan be used as a nutritional supplement to supplement a mammal's dietwith bacterial organisms having the ability to reduce the severity ordevelopment of an autoimmune condition. Examples of mammals include,without limitation, humans, monkeys, dogs, cats, cows, horses, pigs, andsheep.

Any amount of a composition containing P. histicola or P. histicolacomponents can be administered to a mammal. The dosages of P. histicola(e.g., live or killed P. histicola) or P. histicola components candepend on many factors including the desired results. Typically, theamount of P. histicola (e.g., live or killed P. histicola) or P.histicola components contained within a single dose can be an amountthat effectively exhibits anti-inflammatory activity within the mammal.For example, a composition containing live P. histicola can beformulated in a dose such that a mammal receives between about 10³ and10⁸ live P. histicola microorganisms.

The final pH of a composition P. histicola (e.g., live or killed P.histicola) or P. histicola components can be between about 3.5 and about9.5 (e.g., between about 4.0 and about 9.0; between about 4.5 and about9.0; between about 4.5 and about 8.5; between about 5.0 and about 8.5;or between about 6.5 and about 8.0). To obtain such a pH, the pH of thecomposition can be adjusted using a pH-adjusting agent, for example. Itwill be appreciated that pH adjustment can be accomplished with any of awide variety of acids should the composition have a pH that is too high(e.g., greater than 10.0 before adjustment). Likewise, pH adjustment canbe accomplished with any of a wide variety of bases should thecomposition have a pH that is too low (e.g., less than 3.0 beforeadjustment).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Use of P. histicola to Reduce Disease Symptoms in anAnimal Model of Multiple Sclerosis

The following was performed to demonstrate that an ongoing inflammatorycondition such as multiple sclerosis (MS), a demyelinating autoimmunedisease of the central nervous system, can be treated by a systemicanti-inflammatory response induced by P. histicola. Transgenic (Tg) miceexpressing human HLA class II genes (HLA-DR3DQ8) associated with MS canbe used as an animal model to study MS. HLA-DR3DQ8 mice develop severeinflammation and demyelination in CNS mimicking human disease. Asdemonstrated herein, feeding P. histicola to DR3.DQ8.AEo mice afterinduction of experimental autoimmune encephalomyelitis (EAE) reduceddisease incidence and severity. Control bacteria such as C. sputigena orE. coli, however, had not effect on disease incidence or severityindicating that the suppressive effect is unique to P. histicola. Inaddition, P. histicola treated mice exhibited a decrease in myelinantigen specific T cell responses as well as a decrease in the level ofIL-17, an inflammatory cytokine. Treatment with P. histicola alsoresulted in increases in levels of IL-10, an anti-inflammatory cytokine.

Methods Transgenic (Tg) Mice

HLA-DQ8 (DQA1*0103, DQB1*0302), HLA-DR3 (DRB1*0301), and HLA-DR3/DQ8 Tgmice were produced as described elsewhere (Das et al., Hum. Immunol.,61:279-289 (2000); Bradley et al., J. Clin. Invest., 100:2227-2234(1997); and Strauss et al., Immunogenetics, 40:104-108 (1994)). Briefly,HLA class II transgenes were introduced into (B6×SWR)F₁ fertilized eggs.Positive offspring were backcrossed to B10.M mice for severalgenerations. HLA transgenic mice were then mated to class II-deficient(Aβ°) mice and intercrossed to generate the HLA transgenic lines. Togenerate double transgenic mice, single transgenic DR3.Aβ° mice weremated with DQ8.Aβ° Tg lines to produce HLA-DR3/DQ8 Tg lines. These HLAclass-II Tg mice were mated with MHC-II^(Δ/Δ)(AE°) mice (Taneja et al.,J. Immunol., 181:2869-2877 (2008)) to generate AE°.DQ8. AE°.DR3 andAE°.DR3.DQ8 mice. Transgene negative littermates were used as controls.All mice were bred and maintained in the pathogen free environmentaccording to National Institutes of Health and institutional guidelines.All experiments were approved by the institutional committee.

Flow Cytometry

Expression of HLA-DR and HLA-DQ molecules on PBLs, lymph node cells(LNCs), and splenocytes were analyzed by flow cytometry using monoclonalantibodies (mAbs) L227 and IVD12, specific for HLA-DR and HLA-DQ(Lampson et al., J. Immunol., (Baltimore, Md.: 1950) 125:293-299),respectively, as described elsewhere (Bradley et al., J. Clin. Invest.,100:2227-2234 (1997)). Surface expression of CD4 (GK1.5), CD8 (53.6.72),a B cell marker (CD45R (RA3-6B2)), a DC cell marker (CD11c (HL3)), amonocyte/macrophage cell marker (CD11b (M1/70)), and NK cell markers(PK136), CD25 (PC61), CD44 (IM7), and CD45RB (16A)) were analyzed usingfluorescent conjugated mAb from BD Biosciences (San Jose, USA).

Polypeptide

Twenty-amino acid-long synthetic peptide proteolipid protein PLP₉₁₋₁₁₀(YTTGAVRQIFGDYKTTICGK; SEQ ID NO:3; See, GenBank Accession No. NP_000524for full length 277 amino acid PLP polypeptide) was synthesized at thepeptide core facility of Mayo Clinic, Rochester, Minn.

Immunization and T Cell Proliferation Assay

Mice were immunized subcutaneously with PLP₉₁₋₁₁₀ (100 μg) polypeptide,emulsified in CFA (1:1) containing 100 μg of Mycobacterium tuberculosisH37Ra (Difco, Detroit, Mich.) as described elsewhere (Mangalam et al.,J. Immunol., 182: 5131-5139 (2009)). Some immunized mice were sacrificed10 days after immunization, and draining lymph nodes were removed andchallenged in vitro with antigen (Das et al., Hum. Immunol., 61:279-289(2000)). The results are presented as stimulation indices (CPM of testsample/CPM of the control).

Disease Induction

For disease induction, 12-14 weeks old Tg mice were immunizedsubcutaneously in both flanks with 100 μg of PLP₉₁₋₁₁₀ emulsified in CFAcontaining Mycobacterium tuberculosis H37Ra (400 μg/mice) (Mangalam etal., J. Immunol., 182: 5131-5139 (2009)). Pertussis toxin (SigmaChemicals, St. Louis, Mo., USA; 100 ng) was injected i.v. at day 0 and2, post immunization. Mice were observed daily for clinical symptoms,and disease severity was scored as follows: 0, normal; 1, loss of tailtone; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limbparalysis and forelimb paralysis or weakness; and 5, moribundity/death.Mice of both sexes were used.

Treatment with Prevotella or Other Commensal Bacteria

To test therapeutic potential of P. histicola, mice were first immunizedwith myelin antigen (PLP91-110). One week after induction of EAE, micewere treated with bacteria by oral gavage. Commensal Gram negative,anaerobic bacteria (Capnocytophagia sputigena or E. coli) also weretested as a treatment option. DR3DQ8 mice received P. histicola or C.sputigena or E. coli or medium alone starting day 7 post-immunizationand every other day for a total of seven doses. Mice were followed forweight loss, disease incidence, duration, and severity for four weekspost-immunization.

Cytokine Analysis

For cytokine analysis, supernatants from different groups were collectedfrom culture 48 hours after polypeptide stimulation. The concentrationof cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17,GM-CSF, IFN-γ, TNF-α, MCP-1, MIP-1α, MIP-1β, etc.) in the supernatantwas measured using 23-plex BioPlex cytokine bead arrays (BioRad) andsandwich ELISA (TGF-β, IL-22, and IL-23) using pairs of relevantanti-cytokine monoclonal antibodies according to the manufacturer'sprotocol (Pharmingen, San Diego, Calif., USA).

Results

Isolation of Commensal Bacteria and their Effect on PLP₉₁₋₁₁₀ InducedEAE in HLA-DR3.DQ8 Double Transgenic Mice

Commensal bacteria were isolated from small intestinal mucosa of humanpatients and tested their ability to modulate the disease process ofEAE. Prevotella histicola, anaerobic, Gram-negative, non-pigmentedbacteria, were isolated and tested for the ability to modulatePLP₉₁₋₁₁₀-induced EAE in double transgenic mice. EAE was induced inHLA-DR3DQ8 transgenic mice by immunization with PLP₉₁₋₁₁₀ emulsified inCFA at 1:1 ratio. These mice also received pertussis toxin at day 0 andday 2 post-immunization. To test therapeutic potential of Prevotellahisticola, mice were treated with bacteria (oral gavage) 7 day postimmunization. DR3DQ8 mice received either Prevotella histicola or mediumstaring day 7 post-immunization and every other day for a total of 7doses. Mice were followed for weight loss, disease incidence, durationand severity for 4 weeks post-immunization. Capnocytophagia sputigena orE. coli were used as control commensal bacteria.

Prevotella histicola fed mice exhibited significantly reduced diseaseincidence as only 25% (4/20) mice develop EAE as compared 100% diseaseincidence in medium fed (sham treated) DR3DQ8 mice (FIG. 1 and Table 1).Treatment with C. sputigena or E. coli had no effect on development orseverity of disease in DR3DQ8 mice, indicating that only P. histicolahave immunomodulatory effect. No disease was observed in DQ8 singletransgenic mice. Further, disease onset in DR3DQ8 mice in P. histicolatreated group was significantly delayed in bacteria treated mice.

TABLE I Effect of bacteria on PLP₉₁₋₁₁₀ induced EAE in HLA Tg mice^(a)Mean onset of Number of mice with Disease incidence disease ± maximumseverity score Mouse strain (%) SD 1 2 3 4 5 DQ8.AE° 0/10 (0%) — — — — —— (Medium) DR3.DQ8.AE° 20/20 (100%) 10 ± 1 — — 4 10  6 (Medium)DR3.DQ8.AE° 4/20 (25%) 18 ± 2 — 1 3 — — (P histicola) DR3.DQ8.AE° 10/10(100%) 13 ± 2 — 1 5 2 3 (C sputigena) DR3.DQ8.AE° 10/10 (100%) 15.7 ±2.5 — — 6 1 4 (E coli) ^(a)= mice were immunized with 100 μg of PLPpeptide/400 μg Mtb in CFA, and Ptx was administered at 0 and 48 hourspost immunization. Mice were scored daily for disease. The data is fromthree experiments combined.Effect of Prevotella histicola on Antigen Specific T Cell Proliferation

To determine if this protective effect of P. histicola is due todown-regulation of antigen specific T cell responses, splenocytes wereisolated from mice treated with bacteria or medium, and stimulated withPLP₉₁₋₁₁₀ peptide. An antigen specific T cell response was suppressed inDR3DQ8 mice treated P. histicola as compared to sham treated mice (FIG.2).

Effect of P. histicola on Cytokine and Chemokine Production

The levels of cytokine and chemokines between bacteria fed and mediumfed mice were compared to determine if P. histicola protected mice fromEAE by modulating levels of pro and anti-inflammatory chemicalmediators. Splenocytes from bacteria fed mice produced less IL-17, apro-inflammatory cytokine) on stimulation with PLP, while levels ofIL-10, an anti-inflammatory cytokine), were increased (FIG. 3).Surprisingly, levels of IFN-γ were not significantly different betweenthe two groups.

Cells from splenocytes of mice protected from EAE (P. histicola treatedmice) produced reduced levels of MIP-1α, MIP-1β, and MCP-1 as comparedto levels observed in mice with EAE (medium treated) (FIG. 4A). At thesame time, the levels of these chemokines in mesenteric lymph node cellswere significantly higher in protected mice (P. histicola treated mice)as compared to mice with EAE (FIG. 4B).

These results demonstrate that P. histicola can have animmuno-modulatory effect that suppresses proliferation ofIL-17-secreting Th17 cells and increases production of IL-10, animmunoregulatory cytokine. While not being limited to any particularmode of action, P. histicola may modulate EAE in HLA-DR3DQ8 transgenicmice by suppressing production of chemokines in encephalitogenic CD4 Tcells, thereby inhibiting migration of pathogenic cells to CNS.

In another experiment, the frequencies of regulatory T cells (e.g.,CD4⁺FoxP3⁺ regulatory T cells) and tolerogenic dendritic cells (e.g.,CD11b⁺CD11c⁻CD103⁺ tolerogenic DCs) were assessed in the HLA-DR3DQ8transgenic mouse model of EAE using sham treated mice and P. histicolatreated mice. Briefly, AEo.DRB1*0301/DQ8 (HLA-DR3DQ8) Tg mice wereimmunized with PLP₉₁₋₁₁₀ myelin antigen emulsified in CFA. Pertussistoxin was given at day 0 and 2 post-immunization. Seven days postimmunization mice were gavaged on alternate days with either P.histicola (2×10⁹ CFU in 100 μL of TSB culture media) or medium for sevendoses. Mice were monitored daily for development of EAE and scored usingstandard EAE scoring criteria as described elsewhere (Mangalam et al.,J. Immunol., 182(8):5131-9 (2009)). Splenocytes and cells frommesenteric lymph node (MLN) were used for analysis of T regulatory cellsand for analysis of CD11b⁺CD11c⁺CD103⁺ tolerogenic DCs.

A plot was generated from CD4 gated cells, revealing the percentCD4⁺FoxP3⁺ cells in spleen and MLN (FIG. 11). P. histicola treatedHLA-DR3DQ8 mice exhibited an increased frequency of CD4⁺FoxP3⁺regulatory T cells in spleen (80% vs. 24%) and mesenteric lymph node(54% vs. 43%) as compared to the frequencies observed in sham treatedmice (FIG. 11). A plot also was generated from CD11b⁺ and CD11c⁺ gatedcells, revealing the percent CD11b⁺CD11c⁺CD103⁺ cells in spleen and MLN(FIG. 12). P. histicola treated HLA-DR3DQ8 mice exhibited an increasedfrequency of CD11b⁺CD11c⁺CD103⁺ tolerogenic DCs in spleen (22% vs. 6%)and mesenteric lymph node (52% vs. 45%) as compared to the frequenciesobserved in sham treated mice (FIG. 12).

These results demonstrate that treatment with P. histicola leads to anincrease in the frequency of regulatory T cells and tolerogenicdendritic cells. These results also demonstrate that P. histicola canmodulate EAE in DR3DQ8 Tg mice by modulation of a cytokines, regulatoryT cell, and regulatory dendritic cell network. In addition, suppressivedendritic cells can be responsible for conversion of T cells to aregulatory phenotype. The regulatory T cells of treated mice can migratefrom lamina propria to periphery, thus modulating overall immuneresponse.

Example 2 Use of P. histicola to Reduce Disease Symptoms in an AnimalModel of Arthritis

The following was performed to demonstrate that an ongoing inflammatorycondition such as rheumatoid arthritis (RA), a chronic inflammatoryautoimmune disease of the joints, can be treated by a systemicanti-inflammatory response induced by P. histicola. Transgenic (Tg) miceexpressing human HLA class II genes (HLA-DQ8) associated with RA can beused as an animal model to study immunopathogenesis of RA. HLAtransgenic mice expressing HLA-DQ8 (HLA-DQA1*0301/DQB1*0302) were highlysusceptible to collagen-induced arthritis (CIA), an animal model ofhuman RA (Taneja et al. J. Immunol., 181:2869-7 (2008)). As demonstratedherein, feeding P. histicola to DQ8.AEo mice after induction of collageninduced arthritis (CIA) reduced disease incidence and severity. Thegroup of mice receiving medium only exhibited no effect on diseaseincidence or severity indicating that P. histicola has a diseasesuppressive effect. In addition, P. histicola treated mice exhibited adecrease in the level of pro-inflammatory and immunomodulatorycytokines. Treatment with P. histicola also resulted in a decrease inthe levels of anti-type II collagen (CII) specific antibodies.

Transgenic (Tg) Mice

DQ8.Abo mice were generated as described previously. These mice weremated with MHC-II^(Δ/Δ) (AE°) mice (Taneja et al., J. Immunol.,181:2869-2877 (2008)) to generate AE°.DQ8 mice. All mice were bred andmaintained in the pathogen free environment according to appropriateguidelines. All experiments were approved by the institutionalcommittee.

Flow Cytometry

Expression of HLA-DQ molecules on PBLs, lymph node cells (LNCs), andsplenocytes were analyzed by flow cytometry using monoclonal antibodies(mAbs) IVD12, specific for HLA-DQ (Lampson et al., J. Immunol.,(Baltimore, Md.: 1950) 125:293-299), respectively, as describedelsewhere (Bradley et al., J. Clin. Invest., 100:2227-2234 (1997)).Surface expression of CD4 (GK1.5), CD8 (53.6.72), a B cell marker (CD45R(RA3-6B2)), a DC cell marker (CD11c (HL3)), a monocyte/macrophage cellmarker (CD11b (M1/70)), GITR [Glucocorticoid-induced Tumor necrosisfactor (TNF) receptor family-Related] (DTA-1), and CD103 (M290) wereanalyzed using fluorescent conjugated mAb from BD Biosciences (San Jose,USA).

Induction and Evaluation of CIA

Pure native chick type II collagen was obtained by multiple-steppurification as described elsewhere (Griffiths et al., Arthritis Rheum.,24:781-789 (1981)). Tg mice and negative littermates were immunized withchick CII as described elsewhere for CIA the protocol (Taneja et al.,Arthritis Rheum., 56:69-78 (2007)). Mice were monitored for the onsetand progression of CIA from 3 to 12 weeks post-immunization. Thearthritic severity of mice was evaluated as described elsewhere with agrading system for each paw from 0 to 3 (Wooley, J. Exp. Med.,154:688-700 (1981)). The mean arthritic score was determined usingarthritic animals only.

Autoantibodies

Levels of anti-chick and anti-mouse CII IgG Abs were measured in seraobtained 35 days following CII immunization by a standard ELISA and areshown as OD. Briefly, microtiter plates were coatedovernight with CII (6μg/well in KPO₄ (pH 7.6)) at 4° C., washed, and blocked with 1% BSA inPBS/0.05% Tween 20. Sera were added in 4-fold dilution ( 1/100 to1/65,000) and incubated overnight at 4° C. The plates were washed, andperoxidase-conjugated goat anti-mouse IgG (Organon Teknika) was addedfor another overnight incubation at 4° C. After washing,O-phenylenediamine was added, and the colorimetric change was measuredat 410 nm.

T Cell Proliferation Assay

Mice were immunized with 200 μg of CII emulsified 1:1 in CFA (Difco)intradermally at the base of the tail and in one hind footpad. Ten dayspost-immunization, draining lymph nodes/spleen were removed and culturedin vitro. Lymph node cells (LNCs, 1×10⁶) were cultured in HEPES-bufferedRPMI 1640 containing 5% heat-inactivated horse serum and streptomycinand penicillin in 96-well flat-bottom tissue culture plates. Cells werechallenged by adding 100 μL of RPMI 1640 medium (negative control), ConA (20 μg/mL, positive control), and native collagen (50 μg/mL). Todetermine CD4-mediated response, GK1.5 (anti-CD4) Ab was used forblocking. The cells were incubated for 48 hours at 37° C. During thelast 18 hours, the cells were pulsed with [³H]thymidine, and the tritiumincorporation was determined by liquid scintillation counting. Resultsare calculated as A cpm (i.e., mean cpm of triplicate culturescontaining Ag—mean cpm of medium).

Cytokines

Cytokines (IL-1α, IL-1β, IL-5, IL-6, IL-10, IL-12p40, IL-13, IL-17,TNFα, and IFNγ) were measured using the Bio-Plex protein array systemwith the mouse cytokine 23-plex panel as per the manufacturer'sinstructions and analyzed with Bio-Plex manager 2.0 software (Bio-RadLaboratories).

Results

Modulation of Collagen-Induced Arthritis (CIA) by Prevotella histicola.

Immunization of HLA-DQ8.AEo mice with type II collagen (CII) leads todevelopment of collagen-induced arthritis, a model for rheumatoidarthritis (Taneja et al., J. Immunol., 56:69-78 (2007)). Treatment withP. histicola protected DQ8.AEo mice from developing arthritis (FIG. 5).Three groups of mice were included: (1) mice immunized with CII/CFA andtreated with medium, (2) mice immunized with CII and treated with P.histicola, and (3) mice immunized with P. histicola without CII, acontrol group. Ten days post immunization mice were gavaged three timesper week with P. histicola (2×10⁹ CFU in 100 μL of TSB culture media)for up to 7 weeks. Group 1 and group 2 mice were boosted with CII/IFA in6^(th) week. Mice were monitored for arthritis for up to 10 weeks. Miceimmunized with CII and treated with P. histicola exhibited a dramaticdecrease in disease incidence as well as milder disease. Mice only givenP. histicola did not develop any arthritis. Mice receiving medium onlyhad no effect on CIA.

Effect of P. histicola on Auto-Antibodies Levels and T CellProliferation

P. histicola treated mice exhibited reduced humoral antigen-specificresponse. Anti-CII antibodies in sera collected before and aftertreatment with P. histicola exhibited a reduction in antibodies (FIG.6A). Anti-CII antibodies were tested by ELISA. T cell proliferation toCII in vitro did not exhibit any significant reduction in P. histicolavs. medium treated mice. Only P. histicola gavaged mice did not exhibitany antigen (CII)-specific response (FIG. 6B).

Effect of P. histicola on Cytokine Production

All pro-inflammatory and immunomodulatory cytokines produced in responseto CII were reduced in P. histicola treated mice as compared to mediumfed (control) mice (FIG. 7). Cytokines were measured from serum of miceby using multiplex array system.

Effect of P. histicola on Regulatory T Cells and Regulatory DendriticCells (DCs) in Splenocytes and Lamina Propria

Mice treated with P. histicola exhibited a higher number of CD4⁺GITR⁺ Tregulatory cells and CD11c⁺CD103⁺ dendritic cells in splenocytes ascompared to mice receiving medium only (FIG. 8). Treatment with P.histicola also resulted in an increase in regulatory dendritic cells,CD11c⁺CD103⁺ in lamina propria (FIG. 9).

These results demonstrate that P. histicola can have an immunomodulatoryeffect that reduces the disease incidence and severity of CIA in DQ8 Tgmice. While not being limited to any particular mode of action, P.histicola may modulate CIA in DQ8 Tg mice by modulation of cytokines,regulatory T cells, and regulatory dendritic cells network.

In another experiment, regulatory T cell (e.g., CD4⁺FoxP3⁺ regulatory Tcells) and tolerogenic dendritic cell (e.g., CD11b⁺CD11c⁺CD103⁺tolerogenic DCs) responses were assessed in the AEoDRB1*0401/DQ8 mousemodel of arthritis using sham treated mice and P. histicola treatedmice. Briefly, AEoDRB1*0401/DQ8 mice were sham treated or treated withP. histicola three times on alternative days before being immunized with100 μg of type II collagen (CII) emulsified in CFA and four times onalternative days after immunization. Splenocytes were used to isolateDCs (adherent cells) and CD4⁺ T cells by staining with conjugatedantibodies and FAC sorting. Cells were used at 99% purity.

To assess proliferation, CD4⁺ cells from sham treated mice were culturedin vitro with DCs from sham treated or P. histicola treated mice in thepresence or absence of CII. Similarly, CD4⁺ cells isolated from spleensof P. histicola treated mice were cultured with DCs from sham treated orP. histicola treated mice in the absence or presence of CII. A histogramplot was generated from the proliferation results from three mice (FIG.13A). P. histicola treated CD4⁺ cells generated a good T cell responseto CII when it was presented by sham DCs, but not when presented by DCsfrom P. histicola treated mice (FIG. 13A). CD4⁺ cells from sham treatedcultured with DCs from P. histicola treated mice did not show anyproliferation, suggesting P. histicola treated DCs are suppressive (FIG.13A).

The supernatants from the cultures assessed in FIG. 13A were assessed toIL-17 and IL-10 levels. Sham CD4⁺ and DC cultures produced more IL-17than IL-10, while cultures using in vivo P. histicola treated DCscultured with CD4⁺ cells from sham and P. histicola treated miceproduced much higher amounts of IL-10 than IL-17 (FIG. 13B). Theseresults demonstrate that P. histicola treatment can have animmuno-modulatory effect that suppresses proliferation ofIL-17-secreting Th17 cells and increases production of IL-10, animmunoregulatory cytokine. The increase in IL-10 production can be dueto an increase in T regulatory cells or suppressive DCs and may onemechanism of protection.

The frequencies of CD4⁻CD25⁺FoxP3⁺ regulatory T cells in sham treated orP. histicola treated HLA-DRB1*0401/DQ8 mice were assessed. Briefly, micewere treated with P. histicola as described with respect to FIG. 13, andsplenocytes and cells from lamina propria were used for analysis of Tregulatory cells. A plot was generated from CD4⁺ gated cells, revealingthe percent of CD4⁺CD25⁺FoxP3⁺ cells in spleen and lamina propria (FIG.14). P. histicola treated HLA-DRB1*0401/DQ8 mice exhibited an increasedfrequency of CD4⁺CD25⁻FoxP3⁺ regulatory T cells in spleen and laminapropria as compared to the frequencies observed in sham treated mice(FIG. 14). These regulatory T cells may be responsible for the producedIL-10 as shown in FIG. 13B.

These results demonstrate that P. histicola can modulate cytokineproduction in transgenic mice via regulatory T and suppressive dendriticcells. Suppressive dendritic cells can be responsible for conversion ofT cells to regulatory phenotype. The regulatory T cells of treated micecan migrate from lamina propria to periphery, thus modulating overallimmune response.

Example 3 Use of P. histicola and P. histicola Culture Supernatants toReduce Disease Symptoms in an Animal Model if Multiple Sclerosis

The following was performed to demonstrate that P. histicola and P.histicola culture supernatants can modulate EAE. HLA-DR3DQ8 Tg mice wereimmunized with PLP₉₁₋₁₁₀ myelin antigen emulsified in CFA. Pertussistoxin was given at day 0 and 2 post-immunization. Seven days postimmunization mice were gavaged on alternate days either with live P.histicola or culture supernatant of P. histicola or medium only forseven doses. P. histicola was grown in TSB media as described herein,and the culture supernatant of P. histicola was collected bycentrifuging the P. histicola culture. Mice were monitored daily fordevelopment of EAE and scored using standard EAE scoring criteria asdescribed elsewhere (Mangalam et al., J. Immunol., 182(8):5131-9(2009)).

Treatment of DR3/DQ8 mice with media in which P. histicola were culturedresulted in a protective effect with only 50% of mice developing EAEcompared to 100% incidence in control mice (media fed) mice (FIG. 15).P. histicola treated HLA-DR3DQ8 mice were used as positive control andexhibited a strong protective effect (FIG. 15). These results indicatethat live P. histicola and culture supernatants of P. histicola can beused to reduce the severity of symptoms of inflammatory conditions andautoimmune conditions. In addition, these results demonstrate thatculture supernatants of P. histicola may exhibit their immunomodulatoryeffect via products secreted by P. histicola, bacterial lysates presentin the culture supernatant, or both.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A composition comprising: (a) live Prevotellahisticola, dead Prevotella histicola, or a culture supernatant of aPrevotella histicola culture, (b) a buffer, a radical scavenger, anantioxidant, a reducing agent, or a mixture thereof, and (c) apharmaceutically acceptable carrier, wherein said composition is in theform of a pill, a tablet, a powder, a liquid, or a capsule.
 3. Thecomposition of claim 2, wherein said composition comprises said livePrevotella histicola.
 4. The composition of claim 3, wherein saidcomposition contains between 1×10⁷ to 1×10¹¹ cells of said livePrevotella histicola.
 5. The composition of claim 2, wherein saidcomposition comprises said dead Prevotella histicola.
 6. The compositionof claim 5, wherein said composition contains between 1×10⁷ to 1×10¹¹cells of said dead Prevotella histicola.
 7. The composition of claim 2,wherein said composition comprises said culture supernatant of saidPrevotella histicola culture.
 8. The composition of claim 7, whereinsaid culture supernatant was obtained from said Prevotella histicolaculture having greater than 1×10⁷ cells of said Prevotella histicola permL.
 9. The composition of claim 2, wherein said composition comprisessaid buffer.
 10. The composition of claim 2, wherein said compositioncomprises said radical scavenger.
 11. The composition of claim 2,wherein said composition comprises said antioxidant.
 12. The compositionof claim 2, wherein said composition comprises said reducing agent. 13.The composition of claim 2, wherein said composition comprises saidmixture.
 14. The composition of claim 2, wherein said pharmaceuticallyacceptable carrier is a non-aqueous solvent.
 15. The composition ofclaim 14, wherein said non-aqueous solvent is propylene glycol,polyethylene glycol, a vegetable oil, or an organic ester.
 16. Thecomposition of claim 2, wherein said composition is a pill, tablet,powder, or capsule.
 17. The composition of claim 16, wherein saidpharmaceutically acceptable carrier is a non-aqueous solvent.
 18. Thecomposition of claim 17, wherein said non-aqueous solvent is propyleneglycol, polyethylene glycol, a vegetable oil, or an organic ester. 19.The composition of claim 2, wherein said composition is a liquid. 20.The composition of claim 19, wherein said pharmaceutically acceptablecarrier is an aqueous carrier.
 21. The composition of claim 20, whereinsaid aqueous carrier is water, alcohol, saline, or a buffered solution.22. The composition of claim 2, wherein said composition is a pill. 23.The composition of claim 2, wherein said live Prevotella histicola, saiddead Prevotella histicola, or said culture supernatant is encapsulatedin said pill to be released in the intestine of a mammal following oraladministration of said pill to said mammal.
 24. The composition of claim2, wherein said composition is a tablet.
 25. The composition of claim 2,wherein said live Prevotella histicola, said dead Prevotella histicola,or said culture supernatant is encapsulated in said tablet to bereleased in the intestine of a mammal following oral administration ofsaid tablet to said mammal.
 26. The composition of claim 2, wherein saidcomposition is a powder.
 27. The composition of claim 2, wherein saidcomposition a capsule.
 28. The composition of claim 2, wherein said livePrevotella histicola, said dead Prevotella histicola, or said culturesupernatant is encapsulated in said capsule to be released in theintestine of a mammal following oral administration of said capsule tosaid mammal.
 29. The composition of claim 2, wherein said livePrevotella histicola are live cells of the Prevotella histicoladeposited as NRRL accession number B-50329, wherein said dead Prevotellahisticola are dead Prevotella histicola deposited as NRRL accessionnumber B-50329, and wherein said culture supernatant is a culturesupernatant of a culture of the Prevotella histicola deposited as NRRLaccession number B-50329.
 30. The composition of claim 2, wherein the pHof said composition is between about 6.5 and about 8.0.